Para-hydroxystyrene (pHS) is a well-known compound having potential utility in a wide variety of industrial applications, including the production of resins, coatings and inks.
A number of methods for the chemical synthesis of pHS are known. For example, pHS may be produced from ethyl benzene in a five-step process (U.S. Pat. No. 4,503,271) or from para-hydroxyacetophenol in a two step process (U.S. Pat. No. 5,523,378). Although it is possible to generate pHS by these methods, they typically require strongly acidic or basic reaction conditions, high reaction temperature, and generate large amounts of unwanted byproducts. In addition, chemical methods require expensive starting materials, which raise the cost of producing pHS. Despite the wide variety of uses for pHS, an inexpensive source of the material has not been developed.
A number of microorganisms have been found to produce 4-hydroxystyrenes, including fungi (Bayne et al., J. Gen. Microbiol. 95, 188–190 (1976) and Harada et al., J. Gen. Microbiol. 11, 1258–1262 (1976)), yeast (Goodey et al., J. Gen. Microbiol, 128, 2615–620 (1982)), and both Gram-negative and Gram-positive bacteria (Finkle et al. J. Biol. Chem. 237, 2926–2931 (1962), and Lindsay et al., J. Appl. Bacteriol. 39, 181–187 (1975)). In each of the above cases carboxylic acids of the phenylpropanoid class are decarboxylated to produce the corresponding 4-hydroxystyrene.
A para-hydroxycinnamic acid decarboxylase activity converts para-hydroxycinnamic acid (pHCA) to pHS (Hashidoko et al., Biosci. Biotech. Biochem., 58(1), 217–218, (1994)). Release of 4-hydroxystyrene was reported when a DNA fragment carrying the 4-hyroxycinnamate decarboxylase gene from Klebsiella oxytoca was consitutively expressed in E. coli JM109. The recombinant host cell did not contain any additional genes for the production of the para-hydroxycinnamic acid (pHCA), the expected pHS precursor.
Additionally, Hashidoko et al. demonstrated production of hydroxystyrene by decarboxylation of 4-hydroxycinnamic acids by hydroxycinnamate decarboxylase from Klebsiella oxytoca (Arch. Biochem. Biophys. (1998), 359(2), 225–230)). The in vitro biological method of Hashidoko et al. for the production of pHS is limited to very low titer from the expensive starting material, pHCA.
In light of the foregoing, it would be an advancement in the art to provide a method for the production pHS using inexpensive materials such as carbohydrates or sugars and to increase the efficiency of a process for production of pHS through the pHCA intermediate. It would be particularly advantageous if the method produced a high level of the desired product with limited by-products. Development of such a method will require the ability to manipulate the genetic machinery responsible for the conversion of carbohydrates such as glucose to pHCA and pHCA to pHS.
The above mentioned biological and chemical systems provide a number of pathways that may be useful in the production of pHS, however, the efficient production of this monomer has not been achieved. The problem to be overcome therefore is to design and implement a method for the efficient production of pHS from a biological source using an inexpensive substrate or fermentable carbon source. Applicants have solved the stated problem by engineering a microbial host to produce pHS by expressing foreign genes encoding phenylalanine ammonia lyase/tyrosine ammonia lyase (PAL/TAL) and para-hydroxycinnamic acid decarboxylase (PDC) activity.